An LC-MS-based workflow measures the export activity of S1P transporters.

Sphingosine-1-phosphate (S1P) is an active signaling metabolite synthesized by blood cells, exported into blood stream, and can trigger many downstream signaling pathways with disease implications. Understanding how S1P is transported is of great values for dissecting the function of S1P, but most existing methods for measuring S1P transporter activity use radioactive substrates or involve multiple workup steps, hindering their broader uses. In this study, we develop a workflow combining sensitive LC-MS measurement and a cell-based transporter protein system to measure the export activity of S1P transporter proteins. Our workflow demonstrated good applications in studying different S1P transporters SPNS2 and MFSD2B, WT and mutated protein, and different protein substrates. In summary, we provide a simple yet versatile workflow for measuring the export activity of S1P transporters, facilitating future studies of S1P transport mechanism and drug development.